Reference Gene Selection for qRT-PCR Normalization in Iris germanica L.

Quantitative real-time PCR(qPCR)is an effective and widely used method to analyze expression patterns of target genes.Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR.In Iris germanica L.,no studies have yet been published regarding the evaluation of potential reference genes.In this study,nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms(GeNorm,NormFinder,BestKeeper,and RefFinder).The results revealed that ACT11(Actin 11)and EF1α(Elongation factor 1 alpha)were the most stable reference genes in different tissues,whereas TUA(Tubulin alpha)and UBC9(Ubiquitin-protein ligase 9)were the most stable ones in different flower developmental stages.UBC9 and ACT11 were the most stable reference genes in all of the tested samples,while the SAMDC(S-Adenosylmethionine decarboxylase)showed the least stability.Finally,to validate the suitability of the selected reference genes,the relative expression level of IgTPS(beta-caryophyllene synthase)was assessed and highlighted the importance of suitable reference gene selection.This work constitutes the first systematic evaluation of potential reference genes in I.germanica and provides guidelines for future research on gene function and molecular mechanisms on I.germanica and related species.

FRANS HALS德国鸢尾组织培养及植株再生研究

以德国鸢尾(Iris germanica.L)FRANS HALS叶片、球茎作为外植体,利用正交试验设计研究探讨不同激素及不同浓度组合对鸢尾愈伤组织的诱导、不定芽分化以及试管苗生根的影响,为鸢尾快速繁殖、种质资源的保存及利用提供最优的技术方法。结果表明,不同外植体灭菌时间也不同,叶片为3%次氯酸钠3 min,球茎为3%次氯酸钠5 min;鸢尾组培快繁的最适合外植体为球茎;鸢尾愈伤组织诱导和芽分化的最适培养基配方为MS+NAA 0.10 mg/L+6-BA 1.00 mg/L,生根培养基为MS+1\2MS+NAA 0.10 mg/L,试管苗移栽基质采用泥炭∶珍珠岩=8∶2,成活率达90%以上。

红籽鸢尾瞬时表达体系的建立

本试验通过瞬时表达解决红籽鸢尾遗传转化效率低和转化周期长的问题。以红籽鸢尾叶片为材料,用转入植物表达载体pBI121的根癌农杆菌EHA105进行遗传转化。通过研究菌液不同浓度(OD600)、乙酰丁香酮浓度(AS)、侵染时间及共培养时间等因素对红籽鸢尾叶片中GUS基因表达效果的影响。结果表明:在农杆菌浓度OD600为0.6,添加1×10^-4mol/L乙酰丁香酮的侵染液中真空抽吸30 min后,黑暗培养4 d,红籽鸢尾叶片的GUS基因表达效果最好。本试验建立的瞬时表达方法操作简单、转化效率高,为后期红籽鸢尾的基因功能研究及分子育种提供了依据。